A common subunit structure in Clostridium botulinum type A, B and E toxins

doi:10.1016/0006-291X(72)90350-6

Biochemical and Biophysical Research Communications

Volume 48, Issue 1, 11 July 1972, Pages 108-112

https://doi.org/10.1016/0006-291X(72)90350-6 Get rights and content

Abstract

Tryptic activation of $Clostridium botulinum$ type E progenitor toxin of 147,000 mol. wt. involves cleavage of the molecule into at least two polypeptides that are separable when the disulfide bond(s) linking them is reduced. The 50,000 and 102,000 mol. wt. of these chains compare with the 53,000 and 97,000 values of the disulfide-linked polypeptides of type A toxin of mol. wt. 145,000. Type E toxin resulting from trypsinization of its progenitor and naturally activated type A and B toxins have similar subunit structures.

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Cited by (144)

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Botulism is caused by an exotoxin produced by the sporeforming bacterium Clostridium botulinum. Of the seven different toxin types, A, B, and E are the types commonly implicated in foodborne outbreaks of botulism. Clostridium botulinum toxins are extremely potent neurotoxins and generally occur at low concentrations in implicated foods. The detection of C. botulinum and the discrimination of these organisms from other clostridia are based on assays for toxin. This sometimes can be difficult because isolation of pure cultures is rather cumbersome. This article will focus on the detection of botulinum neurotoxins in foods.
Structural insights into the functional role of the Hcn sub-domain of the receptor-binding domain of the botulinum neurotoxin mosaic serotype C/D
2013, Biochimie
Citation Excerpt :
Animals are also susceptible to two BoNT mosaic proteins that are essentially hybrids of the C and D serotypes, BoNT/CD (∼two-third BoNT/C and ∼one-third BoNT/D) and BoNT/DC (∼one-third BoNT/C and ∼two-third BoNT/D). Each BoNT isoform is initially translated as an ∼150 kDa single chain polypeptide that is proteolytically activated and assembled into a molecule with a N-terminal ∼50 kDa light chain (LC) and a C-terminal ∼100 kDa heavy chain (HC) held together with a disulfide bond [8,9]. This mature toxin consists of three modules with distinct functions.
Botulinum neurotoxin (BoNT), the causative agent of the deadly neuroparalytic disease botulism, is the most poisonous protein known for humans. Produced by different strains of the anaerobic bacterium Clostridium botulinum, BoNT effects cellular intoxication via a multistep mechanism executed by the three modules of the activated protein. Endocytosis, the first step of cellular intoxication, is triggered by the ∼50 kDa, heavy-chain receptor-binding domain (HCR) that is specific for a ganglioside and a protein receptor on neuronal cell surfaces. This dual receptor recognition mechanism between BoNT and the host cell's membrane is well documented and occurs via specific intermolecular interactions with the C-terminal sub-domain, Hcc, of BoNT–HCR. The N-terminal sub-domain of BoNT–HCR, Hcn, comprises ∼50% of BoNT–HCR and adopts a β-sheet jelly roll fold. While suspected in assisting cell surface recognition, no unambiguous function for the Hcn sub-domain in BoNT has been identified. To obtain insights into the potential function of the Hcn sub-domain in BoNT, the first crystal structure of a BoNT with an organic ligand bound to the Hcn sub-domain has been obtained. Here, we describe the crystal structure of BoNT/CD–HCR determined at 1.70 Å resolution with a tetraethylene glycol (PG4) moiety bound in a hydrophobic cleft between β-strands in the β-sheet jelly roll fold of the Hcn sub-domain. The PG4 moiety is completely engulfed in the cleft, making numerous hydrophilic (Y932, S959, W966, and D1042) and hydrophobic (S935, W977, L979, N1013, and I1066) contacts with the protein's side chain and backbone that may mimic in vivo interactions with the phospholipid membranes on neuronal cell surfaces. A sulfate ion was also observed bound to residues T1176, D1177, K1196, and R1243 in the Hcc sub-domain of BoNT/CD–HCR. In the crystal structure of a similar protein, BoNT/D–HCR, a sialic acid molecule was observed bound to the equivalent residues suggesting that residues T1176, D1177, K1196, and R1243 in BoNT/CD may play a role in ganglioside binding.
The life history of a botulinum toxin molecule
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There is an emerging literature describing the absorption, distribution, metabolism and elimination of botulinum toxin. This work reveals that the toxin can be absorbed by both the oral and inhalation routes. The primary mechanism for absorption is binding and transport across epithelial cells. Toxin that enters the body undergoes a distribution phase, which is quite short, and an elimination phase, which is comparatively long. During the distribution phase, botulinum toxin migrates to the peri-neuronal microcompartment in the vicinity of vulnerable cells, such as cholinergic nerve endings. Only these cells have the ability to selectively accumulate the molecule. When the toxin moves from the cell membrane to the cell interior, it undergoes programmed death. This is coincident with release of the catalytically active light chain that paralyzes transmission. Intraneuronal metabolism of light chain is via the ubiquitination-proteasome pathway. Systemic metabolism and elimination is assumed to be via the liver. The analysis of absorption, distribution, metabolism and elimination of the toxin helps to create a life history of the molecule in the body. This has many benefits, including: a) clarifying the mechanisms that underlie the disease botulism, b) providing insights for development of medical countermeasures against the toxin, and c) helping to explain the meaning of a lethal dose of toxin. It is likely that work intended to enhance understanding of the fate of botulinum toxin in the body will intensify. These efforts will include new and powerful analytic tools, such as single molecule–single cell analyses in vitro and real time, 3-dimensional pharmacokinetic studies in vivo.
Simultaneous and sensitive detection of six serotypes of botulinum neurotoxin using enzyme-linked immunosorbent assay-based protein antibody microarrays
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Botulinum neurotoxins (BoNTs), produced by Clostridium botulinum, are a group of seven (A–G) immunologically distinct proteins and cause the paralytic disease botulism. These toxins are the most poisonous substances known to humans and are potential bioweapon agents. Therefore, it is necessary to develop highly sensitive assays for the detection of BoNTs in both clinical and environmental samples. In the current study, we have developed an enzyme-linked immunosorbent assay (ELISA)-based protein antibody microarray for the sensitive and simultaneous detection of BoNT serotypes A, B, C, D, E, and F. With engineered high-affinity antibodies, the BoNT assays have sensitivities in buffer ranging from 1.3 fM (0.2 pg/ml) to 14.7 fM (2.2 pg/ml). Using clinical and food matrices (serum and milk), the microarray is capable of detecting BoNT serotypes A to F to similar levels as in standard buffer. Cross-reactivity between assays for individual serotype was also analyzed. These simultaneous, rapid, and sensitive assays have the potential to measure botulinum toxins in a high-throughput manner in complex clinical, food, and environmental samples.
Reversal of BoNT/A-mediated inhibition of muscle paralysis by 3,4-diaminopyridine and roscovitine in mouse phrenic nerve-hemidiaphragm preparations
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Botulinum neurotoxins (BoNTs) comprise a family of neurotoxic proteins synthesized by anaerobic bacteria of the genus Clostridium. Each neurotoxin consists of two polypeptide chains: a 100 kDa heavy chain, responsible for binding and internalization into the nerve terminal of cholinergic motoneurons and a 50 kDa light chain that mediates cleavage of specific synaptic proteins in the host nerve terminal. Exposure to BoNT leads to cessation of voltage- and Ca²⁺-dependent acetylcholine (ACh) release, resulting in flaccid paralysis which may be protracted and potentially fatal.
There are no approved therapies for BoNT intoxication once symptoms appear, and specific inhibitors of the light chain developed to date have not been able to reverse the consequences of BoNT intoxication. An alternative approach for treatment of botulism is to focus on compounds that act by enhancing ACh release. To this end, we examined the action of the K⁺ channel blocker 3,4-diaminopyridine (3,4-DAP) in isolated mouse hemidiaphragm muscles intoxicated with 5 pM BoNT/A. 3,4-DAP restored tension within 1–3 min of application, and was effective even in totally paralyzed muscle. The Ca²⁺ channel activator (R)-roscovitine (Ros) potentiated the action of 3,4-DAP, allowing for use of lower concentrations of the K⁺ channel blocker. In the absence of 3,4-DAP, Ros was unable to augment tension in BoNT/A-intoxicated muscle. This is the first report demonstrating the efficacy of the combination of 3,4-DAP and Ros for the potential treatment of BoNT/A-mediated muscle paralysis.
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A common subunit structure in Clostridium botulinum type A, B and E toxins

Abstract

Biochim. Biophys. Acta

J. Biol. Chem

J. Biol. Chem

J. Biol. Chem

A common subunit structure in $Clostridium botulinum$ type A, B and E toxins